La première partie de ma thèse vise à démêler la structure du complexe NuRD humain. Pour cela, nous avons utilisé la microscopie électronique en transmission (MET), appuyée par la spectrométrie de masse analytique par nos collaborateurs. Nous savions que NuRD était un complexe particulièrement hétérogène et flexible. Nous avons donc fait des efforts particuliers pour développer des protocoles de purification et de réticulation appropriés afin d’obtenir l’échantillon le plus pur et le plus stable possible.
Nous avons purifié NuRD humain endogène à partir de cellules HeLa en utilisant du MBD3 marqué au GFP comme appât pour le complexe complet. Malgré de faibles rendements, nous avons réussi à purifier suffisamment de complexe NuRD humain pour les études de EM.
Les moyennes de classe 2D et les modèles 3D reconstruits à partir de données NuRD humaines montrent une grande hétérogénéité, ce qui rend difficile l'obtention d'une résolution élevée. En conséquence, nous avons décidé de nous concentrer sur le complexe NuRD de Drosophila. Ce complexe est considérablement moins hétérogène et représente une meilleure cible pour les études de EM.
En parallèle, nous avons étudié les interactions de sous-unités individuelles. Nous avons découvert que GATAD2A est capable de se lier à la calmoduline. Cela indique que le calcium peut aider à réguler l'activité de NuRD via la calmoduline.
3.1 Introduction
This chapter focuses on the work towards uncovering the structure of a functional human NuRD complex. For this we used Transmission Electron Microscopy (TEM), a combination of Negative Stain and Cryo-electron microscopy techniques, supported with additional data from Mass Spectrometry.
Classically, X-ray crystallography has been the method of choice to obtain high resolution models of protein structures. Using diffraction patterns of X-rays passing through protein crystals, this technique can resolve the atomic structure of proteins. However, there are serious restrictions to the use of X-ray crystallography. Crystallization of proteins is unpredictable and does not always succeed. More importantly, samples for purification need to be highly homogeneous, which means that different biologically meaningful conformational states of the protein will be excluded from the study. While discrete conformations may be obtained independently from separate crystallography assays, more subtle variations and intermediates are beyond the reach of the technique. Furthermore, crystallization often requires of mutation or complete removal of protein regions prone to disorder, such as intrinsically disordered domains, and the crystallization conditions may lead to structures of conformations of the studied protein which are not relevant for its function. These restrictions are particularly relevant for macromolecular protein complexes which often rely on conformational and compositional plasticity to fulfil their functions.
Cryo-EM and state of the art image processing, with its own strengths and weaknesses, provides an alternative approach to the study of protein complex structure at high resolution, and hence was chosen for this study.
3.2 Methods
3.2.1 Cell culture
Mammalian cell cultures: The endogenous human NuRD was produced using a stable HeLa cell line generated by our collaborator Michiel Vermeulen, based on a previously described construct (Kloet et al., 2014). Briefly, HeLa cells were transfected with a N-terminally tagged, doxycycline inducible MBD3 construct using Lipofectamine (Invitrogen). The construct was created by cloning the tandem tag, comprising a enhanced GPF, two Tobacco Etch Virus (TEV) protease cleavage sites and decahistidine into a pcDNA5 vector, using a flippase – flippase recognition target (flp-FRT) recombination system. Then MBD3, amplified with
BamHI and XhoI, was inserted into the cloning site of the vector. The vector also
included genes for blasticidin and hygromycin antibiotic resistances. Cell stocks frozen in presence of DMSO were thawed and plated in 100mm diameter plates in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS) and L-glutamine and in presence of penicillin and streptomycin antibiotics. After 16-24 hours of growth confluent plates where lightly treated with trypsine to harvest cells. These where plated again in 150 mm diameter plates with hygromycin and blasticidin antibiotics to select for the desired construct. After three days the plates showing 70-90% confluence where induced during 16 hours using doxycycline in order to produce recombinant GFP-MBD3 in amounts similar to endogenous MBD3 protein levels (Kloet et al., 2014, Baymaz et al., 2014). Cells where collected and cytosolic and nuclear extracts obtained as described before with minimal modifications (Kloet et al., 2014). After 5 minutes exposure to trypsin at room temperature to dislodge them from the plate, pelleted by centrifugation at 500 g during 5 minutes at 4°C and washed in PBS. Following collection the cells were incubated in buffer A [10 mM HEPES at pH 7.9, 10 mM KCl, 1.5 mM MgCl2
and 0.15% NP40, complemented with C protease inhibitor tablets (Roche)] and lysed using a dunce homogeniser with a type B pestle (tight). Cytosolic extracts where separated from the nuclei and the membrane lipids by centrifugation at
3200 g during 15 minutes at 4°C. Nuclei where further incubated during 30 minutes in buffer B (20 mM HEPES at pH 7.9, 420 mM NaCl, 2mM MgCl2 0.5 mM EDTA, 20% glycerol, 0.1% NP40 and 0.5 mM DTT) to lyse them. Both cytosolic and nuclear extracts were ultracentrifuged at 20000 g for 20 minutes at 4°C and frozen at -80°C for later use.
Insect cell culture: Individual hNuRD subunits were expressed in a Spodoptera
frugiperda (Sf 21) (Invitrogen) cell line using the MultiBac baculovirus – insect cell
expression system (Berger et al., 2004). The insect cells were transfected with a recombinant bacmid encoding for either wild type GATAD2A and CBP-tagged CHD4 or wild type GATAD2A alone. Sequences encoding these proteins were provided by our collaborators in the Vermeulen group. The first virus generation (V0) was produced in 3 ml cultures in a 6-well plate. This V0 was used to infect 25 ml Sf21 cultures in shaker flasks, producing the V1 generation of viruses. This V1 was finally used for protein production by infection of 400 ml of Sf21 insect cells in Sf-900 medium in 2 litre Erlenmeyer flasks which were incubated at 25°C orbiting at 80 rpm (Corning). The cells were harvested after 72 to 96 hours, after the day of proliferation arrest (DPA). DPA was defined by the plateau of the expression of the the YFP internal expression reporter which is encoded by the baculovirus. YFP levels were determined each 24h to to follow recombinant protein expression. The cells were harvested by centrifugation at 800 x g in a JA8.1000 rotor for 10 min at 4° C. Cells were lysed using short bursts of sonication for a total of 150 seconds at 4°C. Cytosolic extracts were separated by centrifugation at 1700 g for 20 minutes. GATAD2A and CHD4 were co-expressed in insect cells using the MultiBac system (Berger et al., 2004), following the same procedure described above. The baculovirus construct encoding wild type GATAD2A and CBP-tagged CHD4 was provided by our collaborators.
3.2.2 Protein biochemistry
Protein purification from mammalian cells: The endogenous NuRD from HeLa
cells was purified as previously described (Kloet et al., 2014). After purification via GFP affinity chromatography I eluted the complex by cleavage using Glutathione S-Transferase (GST) tagged TEV protease in TEV cleavage buffer (10 mM HEPES at pH 8, 150 mM NaCl, 1 mM EDTA, 3% glycerol, 0.025% NP40 and 1mM DTT). The TEV protease was eliminated by a GST resin affinity purification step. Subsequently, I concentrated the protein and stabilized the hNuRD complex by Bissulfosuccinimidyl Suberate (BS3) cross-linking. BS3 (Thermo Fisher Scientific) is a lysine-specific cross-linking agent with a linker length of 11.5 Å. The final purification step comprised size exclusion chromatography using an Akta Micro and a Superose 6 column (GE Healthcare Life Sciences). Following this protocol I obtained a sample that showed clearly all bands for the known hNuRD subunits on a Coommassie-stained SDS gel. Subsequent optimization of the incubation times and elution volumes allowed improved protein yields. Despite this improvement, the yields remained low. Purification of 30 ml of nuclear extracts, adjusted to a protein concentration of approximately 6mg/ml, would lead to 600 μL of sample, which would then be concentrated (either by osmotic concentration inside a membrane using highly concentrated polyethylen glycol to extract buffer from the sample, or with 10kDa Amicon concentration 4ml columns) up to 10 times to a final concentration of 0.1-1 mg/ml. The GRAFIX (Gradient Fixation) procedure (Kastner et al., 2008), when applied, consisted in gradient centrifugation in a glycerol gradient in the presence of mild glutaraldehyde crosslinking. About 50-100 μL complex was loaded onto a 4ml centrifuge tube (Beckman 7/16x2–3/8 P.A) containing a gradient of 10-30% v/v glycerol and 0-0.15% glutaraldehyde in the same buffer containing the sample (10 mM HEPES at pH 8, 150 mM NaCl, 1 mM EDTA, 3% glycerol, 0.025% NP40 and 1mM DTT), generated with a Gradient Master device (Biocomp/Wolf laboratories) and centrifuged at 4°C, 34,000 rpm for 18 h in a SW60Ti rotor (Beckman). Centrifuged samples were fractionated into 0.2 ml aliquots using a peristaltic pump (Biorad). The cross-linking activity of
glutaraldehyde was quenched with 2 μl of 80 mM glycine pH 7.6 immediately after fractionation.
Protein purification from insect cells; The cytosolic extracts obtained from the
insect cells where subjected to affinity chromatography using calmodulin-agarose resin (Sigma-Aldrich). The extracts where incubated in the resin in a calcium containing binding buffer (50 mM TRIS at pH 8, 300 mM NaCl, 1 mM MgCl2 and 2 mM CaCl2), extensively washed and eluted with three applications of elution buffer containing EGTA (50 mM TRIS at pH 8, 300 mM NaCl, 1 mM MgCl2 and 2 mM EGTA). EGTA was used instead of the more common EDTA chelating agent to avoid depleting the magnesium ions in the buffer, as EGTA shows higher affinity for calcium. Elutions were pulled and concentrated two times with Amikon 10kDa pore size 4ml concentrating columns. were The purification protocol was optimised by increasing the elution time (from 5 minutes to 30 minutes each elution) and increasing the concentration of the EGTA in the elution buffer (along with an increase of magnesium chloride in the buffer, to 3mM each).
After affinity chromatography the co-expressed wild type GATAD2A and CBP tagged CHD4 were subjected to size exclusion chromatography using a 10-30% glycerol gradient generated with a Gradient Master device (Biocomp/Wolf laboratories) in elution buffer without EGTA and with additional 1mM ZnCl2, loaded in a centrifuge tube (Beckman 7/16x2–3/8 P.A) and centrifuged at 4°C, 34,000 rpm for 16 h using a SW60Ti rotor (Beckman). Fractions of 0.2 ml were collected using a peristaltic pump (BioRad). This sample and the pure wild type GATAD2A were also subjected to analytical size exclusion chromatography using an Akta Micro and a Superose 6 column (GE Healthcare Life Sciences). Wild type GATAD2A was also incubated for 2h with 0.025 mg/ml (final concentration) of calmodulin (CaM) (Sigma Aldrich) before loading into the Superose 6 column to address the possible binding between the two molecules.
3.2.3 Electron microscopy
Grid preparation: 5 μl of sample was adsorbed onto carbon film for 60 s and
blotted by gentle application of blotting paper (Whatman, Grade 1). Afterwards the carbon film was floated into 1% uranyl acetate staining for 30 s and deposited on copper 300 mesh grids (Electron Microscopy Sciences, EMS300-Cu). The graphite carbon film was prepared in house by evaporating carbon over a mica plastic support. We used carbon rods and the thickness of the carbon film (usually between 1 and 50 nm) was controlled by exposure time and visually checked in the microscope, but not directly measured for each preparation.
F20 data collection: In collaboration with Dr. Manikandan Karuppasamy from our
lab, I used the F20 microscope at the Institut de Biologie Structurale (IBS, Grenoble GIANT campus) at 80 kV and 40kx magnification (2.85Å/pixel) and collected 250 tilt pairs images (45° tilt) for random conical tilt (RCT) reconstruction (Radermacher, 1988). Moreover, 300 untilted micrographs were collected for refinement of the initial volumes.
Image processing: Particle picking from the 250 micrograph pairs resulted in
39,361 particle pairs. We used the untilted images for iterative 2D Multivariate Statistical Analysis (MSA) and classification resulting in 750 2D class averages. For this, I used the iterative MSA, classification and Multi-Reference-Alignment (MRA) implemented in the IMAGIC-5 software (Van Heel & Keegstra, 1981). 407 of 750 2D class averages were used to start the RCT reconstructions in XMIPP 1 (Marabini et al., 1996). The resulting 407 different volumes were averaged into 25 volumes using ML-tomo in XMIPP 1. We used the most populated 6 volumes as input models for further refinement of the structures with XMIPP 1 and RELION 1.3 (Scheres, 2012) using 65,134 additional untilted particles. The classes showing best results were subjected to further classification and gold standard refinement processes with RELION 1.3.
3.3 Results
3.3.1 Purification and EM analysis of endogenous NuRD
I purified human NuRD complex (hNuRD) from a stable HeLa cell line received from our collaborator Michiel Vermeulen (University of Njimegen). I followed an adapted version of the published protocol (Kloet et al., 2014) and was helped by Alice Aubert from the Berger group (EMBL Grenoble). These HeLa cells carry a GFP-tagged recombinant version of MBD3 which is used as a bait for affinity purification, as described in the methods part. A double TEV cleavage site allow for easy elution from the GFP trap (Figure 3.1a). Initial purifications showed a yield comparable to that previously reported by Dr. Vermeulen (Figure. 3.1b and c). Small adjustments to incubation times and volumes in the protocol allowed considerable improvement of the yields (Figure 3.1d), but these remained considerably low (needing, after optimization, 200 to 400 150mm culture plates for each purification, yielding between 50-200 μl of purified sample at 0.1-1 mg/ml protein concentration). All the proteins known to form the core hNuRD complex were easily detected on the Coommassie stained SDS-PAGE gels with the exception of DOC1 which has a molecular weight of 12kDa. This protein is very small compared to the other subunits of the NuRD complex, which makes its observation in co-purification experiments rather difficult. However, DOC1’s presence in the complex has been previously documented by mass spectroscopy experiments (Kloet et al., 2014). The presence of the hNuRD complex subunits was further confirmed by Western blotting, using specific mouse monoclonal antibodies, which were provided by our collaborators in the Laue lab (Figure 3.1e). The presence of DOC1 could not be confirmed due to the lack of monoclonal antibodies against this protein. In order to stabilise the hNuRD complexes and reduce their heterogeneity I performed cross-linking by GRAFIX (Kastner et al., 2008) and by the lysine specific cross-linker BS3. GRAFIX involves gradient centrifugation of the protein in addition to cross-linking with glutaraldehyde, but also results in dilution of low concentration samples. Due to the naturally low levels
Figure 3.1: Purification of hNuRD A) Cartoon of the MBD3 construct used as
bait for the purification of full size endogenous hNuRD complex. For affinity purification we used anti GFP antibody fragments immobilised on agarose beads. The MBD3 construct comprises a recombinant MBD3 tagged with N-terminal GFP. A double TEV cleavage site allows removal of the GFP. B) This gel shows hNuRD complex purified in his lab. All subunits except DOC1 are visible. C) Early purifications of hNuRD complex in our lab showed similar quality to that obtained by Dr. Vermeulen. The gel in C was loaded with approximately half the sample volume of the one in B. D) After protocol optimization the sample quality improved. All subunits except DOC1 are visible and different isoforms are easily distinguishable. The volume loaded in this gel was approximately a quarter of that loaded in the gel in B. E) Wester blots showing the purified hNuRD subunits (blots provided by Alice Aubert). MWM (molecular weight marker) units shown in kDa.
of endogenous hNuRD synthesis cross-linking with a highly concentrated BS3 solution proved to be more effective in this case. To further purify the BS3 cross-linked complexes and to prevent aggregates and the cross-linking of multiple complexes rather than intra-complex a size exclusion chromatography step was added to the BS3 cross-linking protocol. A Superose 6 size exclusion column and the Akta micro system was used for this step. As a result I obtained pure hNuRD complexes in sufficient quantity for EM studies.
3.3.2 First hNuRD models by random conical tilt
In collaboration with Dr. Manikandan Karuppasamy, I collected negative stain tilted micrograph pairs from the hNuRD sample. The micrographs obtained show clear individual particles at good concentrations (Figure 3.2a), but they also show particle heterogeneity, despite our cross-linking efforts aimed at minimising this
Figure 3.2: Negative stain EM of the hNuRD complex. A) A negative stain
micrograph of the hNuRD complex, obtained with an F20 microscope (FEI) at 40.000 x magnification. The scale bar is 25 nm long. The micrograph shows similar sized, well defined and abundant particles, albeit heterogeneous. B) The hNuRD 2D class averages corroborate the initial observations. The particles are well defined but heterogeneous. The average size of the particles is 20 nm, but globular and elongated shapes can be observed.
Figure 3.3: First 3D reconstructions of the hNuRD complex. A)The most
populated six volumes resulting from the XMIPP RCT procedure are shown in different colours. The top left and bottom left volumes appear more defined than the rest and were used for further processing. B and C) The left hand models were further processed in RELION by 3D classification and 3D refinement, to a resolution of 32 and 35 Å respectively. Front and back views are shown for each volume. Next to the3D reconstructions a comparison of their 2D projections and previously obtained reference free 2D class averages is shown. Good agreement between these suggests that the processing is consistent.
known characteristic of the complex. Preliminary 2D processing of the particles using IMAGIC-5 (Van Heel & Keegstra, 1981) and RELION 1.3 (Scheres, 2012) confirmed this heterogeneity (Figure 3.2b). I picked tilt pairs from these micrographs and used them to build the first 3D models of hNuRD using the Random Conical Tilt RCT procedure (Radermacher, 1988). This procedure relies on the fact that randomly oriented particles showing the same face in a micrograph will expose different faces when the grid is tilted. Using the untilted images of the particles these can be classified and oriented. The known tilt angle of the corresponding tilted pairs can be used to back project the images into a 3D volume. This back projection (called “back” as opposed to the projection of a view from a 3D volume into a 2D image) is possible thanks to a property of the Fourier transform, which states that the Fourier transform of a 2D projection of a 3D volume will match the central slice of the 3D Fourier transform of said volume which is perpendicular to the projection angle. From the 25 volumes obtained by this procedure I selected the most populated 6 volumes for further processing.